During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below, despite the disruption of relocating the lab at the end of the calendar year. We have assisted our collaborators by providing materials and cell lines, resulting in the publication of several manuscripts. We continued to mentor and teach, both standard and feeder-free, pluripotent stem cell cultures, provided assistance and advice on the generation of human induced pluripotent stem cells (hiPSCs) from collaborators samples, as well as assistance and advice on differentiation strategies as requested. We have also been evaluating nave pluripotent culture protocols, which could be important in studying diseases related to the X chromosome, and developing simple assays to assess the naivety of the culture. In terms of bringing pluripotent stem cells to the clinic, we have provided advice on assays and culture of iPSCs related to Dr. Kapil Bhartis (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. We also continue to advise Dr. Melvin L. DePamphilis (NICHD) on the study of the role of geminin (GMNN) in controlling mouse and human pluripotent stem cell growth and differentiation. Traceable markers in human pluripotent stem cell are a valuable tool to study biological processes. To generate various human pluripotent stem cell clones expressing tagged proteins, CRISPR technology has been adapted to introduce GFP or RFP cassettes to the C-termini of endogenous proteins. To test the validity of the system, an EGFP-2A-PuroR cassette has been successfully introduced to the nucleolar protein, fibrillarin (FBL) in a human iPSC line. The targeted cells show strong fluorescence staining in their nucleoli, confirming successful tagging. Tagging of other proteins of interest is being planned. In collaboration with Dr. Curtis Harris (NCI), a knock-out project has been initiated where the expression of a specific variant of p53 will be specifically disrupted while the normal p53 expression remains intact. Sequence analysis has confirmed successful substitution as designed and the expression pattern of various p53 proteins are being analyzed. In another collaboration with Dr. Harris, we are carrying out experiments to address genomic instability in human pluripotent stem cells under long-term cell culture conditions, particularly the role of p53 in controlling human pluripotent stem cell growth, differentiation, and the maintenance of genomic stability in these cells. In collaboration with Dr. Ettore Appella (NCI), we are also investigating the role of p53 in the regulation of the self-renewal and differentiation of neural precursors or neural stem cells. Spontaneous differentiation of Wip1- and p53-knockout-derived neurospheres toward neuronal precursors suggests an important role for Wip1 and p53 in stem cell survival, cellular differentiation, and genomic stress response. Our data also provide critical insights into the genomic-stress management in hPSC culture and differentiation. In collaboration with Dr. Anton Jetten (NIEHS), the role of GLI-similar 3 (GLIS3) is being investigated. Deficiency in GLIS3 causes many pathological conditions including neonatal diabetes and congenital glaucoma. Previously, we generated human embryonic stem cell (hESC) lines in which the GLIS3 gene was knocked out using CRISPR/Cas9 technology. To study the role of GLIS3 in normal physiological conditions, new hESC clones are being generated in which GLIS3 protein is HA tagged with GFP expressed from the same endogenous promoter. In a second study, another member of the GLI-similar gene family, GLIS2, which encodes a zinc finger protein, is being investigated. This protein is suggested to have a role in the regulation of kidney morphogenesis with mutations in the gene causing nephronophthisis (NPHP), an autosomal recessive kidney disease. To study the function of GLIS2 protein, the GLIS2 gene has been knocked out using CRISPR technology. In vitro differentiation studies using GLIS2-deficient hESC lines are under way. In collaboration with Wei Li (NEI), we published our completed studies on iPSCs derived from a hibernator, 13-lined ground squirrel, as a platform for studying cold adaptation (Ou, J. et al. Cell 2018 173(4):851-86). In collaboration with Dr. Pamela Robey (NIDCR), we have analyzed the advantages and limitations of current mouse genetic tools widely used to understand skeletal and hematopoietic stem cell niches in bone marrow. Many experimental designs have unavoidable technological limitations and bias, leading to experimental discrepancies, data reproducibility issues, and frequent data misinterpretation. Consequently, there are conflicting views relating to fundamental biological questions, including origins and locations of skeletal and hematopoietic stem cells in the bone marrow. In a published article (Chen KG et al., Stem Cell Reports, 2017 9: 1343-1358), we systematically unraveled complicated data interpretations via comprehensive analyses of technological benefits, pitfalls, and challenges in frequently used mouse models and discuss their translational relevance to human stem cell biology. Particularly, we emphasized the important roles of using large human genomic data-informatics in facilitating genetic analyses of mouse models and resolving existing controversies in mouse and human bone marrow stem-cell biology. In collaboration with Dr. Wei Zheng (NCATS) and Dr. Michael Gottesman (NCI), we discussed the use of human pluripotent stem cells (hPSCs) and their differentiated derivatives in drug discoveries, in order to define a pathway to implement hPSC-based drug discovery (hPDD). In a recent publication (Chen KG et al., Trends Mol Med. 2018 pii: S1471-4914(18) 30136-9), we dissected representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discussed mechanisms of drug discovery and drug repurposing, and the roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis. As always, we update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.